Method for detecting ocln-arhgap26 gene

ABSTRACT

The object of the invention is to elucidate a new causative gene of cancer, polynucleotide, and thereby provide a method for detecting the polynucleotide or a polypeptide that is encoded by the polynucleotide, as well as a primer set or a detection kit for such detection. The detection method detects a fusion gene of a part of an OCLN gene and a part of an ARHGAP26 gene, or a fusion protein encoded by such gene. The primer set includes a sense primer designed from a section encoding OCLN and an antisense primer designed from a section encoding ARHGAP26.

This application is a continuation of U.S. patent application Ser. No.15/753,775 filed Feb. 20, 2018, which is a national stage filing under35 U.S.C. § 371 of PCT/JP2016/074440, filed Aug. 23, 2016, which claimspriority to U.S. Provisional Patent Application No. 62/209,095, filedAug. 24, 2015, and Japanese Patent Application No. 2015-250996, filedDec. 24, 2015, the contents of which are each incorporated herein byreference in their entireties.

This application incorporates-by-reference the sequence listingcontained in the text file named 127206_05703_Sequence_Listing.txt,which was created on Mar. 11, 2020 and is 17,458 bytes in size.

TECHNICAL FIELD

The present invention relates to a method for detecting a novel fusiongene.

BACKGROUND ART

Occludin (OCLN) gene exists on the long arm of human chromosome 5, and aprotein encoded by this gene is a four transmembrane protein. OCLN formsa complex with a claudin family protein that is also a fourtransmembrane protein and constitutes a tight junction (J Cell Biol.1998; 143(2): 391-401) and enhances electric resistance between cells byover-expression of OCLN in cells (J Cell Sci. 1996; 109: 2287-2298). Assuch, OCLN is considered to hold a role in the barrier function of tightjunction. With regards to cancer, over-expression of OCLN in cells isreported to enhance the apoptosis signal and to suppress metastaticpotential (Cancer Res. 2006; 66(18): 9125-9133).

Rho GTPase activating protein 26 (ARHGAP26) gene, which has GTPaseactivating function exists on the long arm of human chromosome 5, sameas OCLN, and the protein encoded by this gene is a GTPase activatingprotein possessing a Rho-GAP domain at the center. ARHGAP26 gene isknown to have a function to enhance the GTP hydrolase activity of thesmall GTPase protein family, particularly RhoA and CDC42 (J Biol Chem.2000; 275(49): 38605-38610.). With regards to cancer, a fusion gene withclaudin 18 (CLDN18) gene was found in 3 to 15% of patients suffered fromdiffuse type gastric cancer (Nature 2014; 513(7517): 202-209, Cell Rep.2015; 12(2): 272-285), and a fusion gene with a mixed-lineage leukemia(MLL) gene was found in leukemia patients (Proc Natl Acad Sci USA. 2000;97(16): 9168-9173., Genes Chromosomes Cancer 2004; 41(4): 400-404).

There are no reports so far of a fusion gene composed of OCLN andARHGAP26.

SUMMARY OF INVENTION Problem to be Solved by Invention

The present invention aims to elucidate a polynucleotide as a novel generesponsible for cancer, and thereby to provide a method for detecting apolynucleotide or a polypeptide that is encoded by the polynucleotide,as well as a primer set or a detection kit for such detection.

Means for Solving the Problems

The present inventors isolated and identified a novel fusion gene from astomach cancer cell line, in which a part of the ARHGAP26 gene and apart of the OCLN gene are fused together (Example 1), and found thatthis fusion gene was the causal cancer gene by the fact that theviability of the stomach cancer cell line declined with the suppressionof expression of fusion genes in the stomach cancer cell line thatendogenously expresses such fusion genes (Example 2, Example 4). Thepresent inventors constructed a detection method of a fusion gene basedon these findings, and provided primer sets for such purpose, therebyusing the detection of such fusion gene made it possible to selectcancer patients (particularly, stomach cancer patients) that testpositive for a fusion gene composed of an OCLN gene and an ARHGAP26 gene(Example 3).

In other words, the present invention relates to [1] to [24] shownbelow.

[1] A method for detecting a fusion gene composed of an occludin (OCLN)gene and a Rho GTPase activating protein 26 (ARHGAP26) gene, wherein themethod comprises a step of detecting whether a polynucleotide thatencodes a polypeptide described by either (1) or (2) shown below existsin a sample obtained from a subject:

(1) a polypeptide that comprises an amino acid sequence having no lessthan 90% identity with an amino acid sequence represented by SEQ ID NO:2;

(2) a polypeptide that comprises an amino acid sequence represented bySEQ ID NO: 2, or a polypeptide that comprises an amino acid sequencerepresented by SEQ ID NO: 2, in which 1 to 10 amino acids are deleted,substituted, inserted and/or added.

[2] The method according to [1], wherein the polypeptide comprises anamino acid sequence having no less than 90% identity with an amino acidsequence represented by SEQ ID NO: 2, and has an ability to developtumor.

[3] The method according to [1], wherein the polypeptide comprises anamino acid sequence represented by SEQ ID NO: 2 and has an ability todevelop tumor, or the polypeptide comprises an amino acid sequencerepresented by SEQ ID NO: 2, in which 1 to 10 amino acids are deleted,substituted, inserted and/or added, and has an ability to develop tumor.

[4] The method according to [1], wherein the polypeptide consists of anamino acid sequence represented by SEQ ID NO: 2.

[5] A method for detecting a fusion gene composed of an OCLN gene and anARHGAP26 gene comprising a step of detecting whether a polynucleotidethat encodes a polypeptide consisting of an amino acid sequencerepresented by SEQ ID NO: 2 exists in a sample obtained from a subject.

[6] The method according to any one of [1] to [5] further comprising astep in which it is judged when a polynucleotide targeted in detectionis detected, that a fusion gene composed of an OCLN gene, and anARHGAP26 gene exists.

[7] The method according to any one of [1] to [6], further comprising astep of amplifying a nucleic acid existing in a sample obtained from asubject, or a step of hybridizing a probe with a nucleic acid existingin a sample obtained from a subject to detect a polynucleotide targetedin detection.

[8] The method according to [7] comprising a step of amplifying thenucleic acid existing in a sample obtained from a subject using a primerset shown below:

a primer set for detecting a fusion gene composed of an OCLN gene and anARHGAP26 gene, the primer set comprising a sense primer designed from asection encoding OCLN and an antisense primer designed from a sectionencoding ARHGAP26, wherein the antisense primer consists of anoligonucleotide that hybridizes under a stringent condition with apolynucleotide targeted in detection, and the sense primer consists ofan oligonucleotide that hybridizes under a stringent condition with acomplementary strand of a polynucleotide targeted in detection.

[9] The method according to [8], wherein the sense primer consists of anoligonucleotide that hybridizes under a stringent condition with acomplementary strand of a polynucleotide consisting of base no. 1 to 891of SEQ ID NO: 1, and the antisense primer consists of an oligonucleotidethat hybridizes under a stringent condition with a polynucleotideconsisting of base no. 892 to 2064 of SEQ ID NO: 1.

[10] The method according to any one of [7] to [9] comprising a step ofamplifying the nucleic acid existing in a sample obtained from a subjectusing a primer set shown below:

a primer set for detecting a fusion gene composed of an OCLN gene and anARHGAP26 gene, wherein a sense primer consists of an oligonucleotide ofat least 16 random consecutive bases between base no. 1 to 891 of SEQ IDNO: 1, and an antisense primer consists of an oligonucleotidecomplementary to an oligonucleotide of at least 16 random consecutivebases between base no. 892 to 2064 of SEQ ID NO: 1.

[11] The method according to any one of [7] to [10] further comprising astep of detecting whether an amplified nucleic acid fragment of a targetsize was obtained.

[12] The method according to [11] further comprising a step in which itis judged when an amplified nucleic acid fragment of a target size isobtained, that a fusion gene composed of an OCLN gene and an ARHGAP26gene exists.

[13] The method according to any one of [7] to [10] further comprising astep of determining a base sequence of an amplified nucleic acidfragment.

[14] The method according to [13] further comprising a step in which itis judged when an amplified nucleic acid fragment includes a basesequence of a section encoding OCLN and a base sequence of a sectionencoding ARHGAP26 in a same fragment, that a fusion gene composed of anOCLN gene and an ARHGAP26 gene exists.

[15] The method according to [7] comprising a step of hybridizing aprobe with the nucleic acid existing in a sample obtained from asubject, wherein the probe comprises an oligonucleotide that hybridizeswith the polynucleotide under a stringent condition.

[16] The method according to [15] comprising a step of performing insitu hybridization using a sample obtained from a subject, a probedesigned from a section encoding OCLN of the polynucleotide, and a probedesigned from a section encoding ARHGAP26 of the polynucleotide.

[17] The method according to [16] using multiple types of probesdesigned from a section encoding OCLN, and multiple types of probesdesigned from a section encoding ARHGAP26.

[18] The method according to [7], [16] or [17] using multiple types ofadjacent probe pairs comprising an oligonucleotide that is complementaryto an oligonucleotide of at least 16 random consecutive bases betweenbase no. 1 to 891 of SEQ ID NO: 1, and multiple types of adjacent probepairs comprising an oligonucleotide that is complementary to anoligonucleotide of at least 16 random consecutive bases between base no.892 to 2064 of SEQ ID NO: 1, in a step of hybridizing a probe with thenucleic acid existing in a sample obtained from a subject.

[19] The method according to any one of [16] to [18] further comprisinga step of amplifying hybridization signals.

[20] The method according to any one of [16] to [19] further comprisinga step of detecting a signal overlap of a signal from a probe designedfrom a section encoding OCLN and a signal from a probe designed from asection encoding ARHGAP26.

[21] The method according to [20] further comprising a step in which itis judged when two signals are detected at a same position, that afusion gene composed of an OCLN gene and an ARHGAP26 gene exists.

[22] The method according to any one of [1] to [21] comprising a step ofobtaining a sample from a subject.

[23] The method according to any one of [1] to [22], wherein the subjectis a cancer patient.

[24] The method according to [23], wherein cancer is stomach cancer.

Further, the present invention relates to [25] to [27] shown below.

[25] A method for detecting whether cancer exists in a subjectcomprising the step according to any one of [1] to [21].

[26] The method according to [25] comprising a step of obtaining asample from a subject.

[27] The method according to [25] or [26], wherein cancer is stomachcancer.

Further, the present invention relates to [28] to [32] shown below.

[28] The method for diagnosing cancer in a subject comprising a stepaccording to any one of [1] to [21].

[29] The method according to [28] comprising a step of obtaining asample from a subject.

[30] The method according to [28] or [29] further comprising a step inwhich it is judged when a fusion gene composed of an OCLN gene and anARHGAP26 gene is detected in a sample obtained from a subject, thatthere is a high possibility of the subject having cancer.

[31] The method according to [28] or [29], wherein cancer is stomachcancer.

[32] The method according to [29] further comprising a step in which itis judged when a fusion gene composed of an OCLN gene and an ARHGAP26gene is detected in a sample obtained from a subject, that there is ahigh possibility of a subject having stomach cancer.

Further, the present invention relates to [33] to [36] shown below.

[33] A method for identifying a subject that is a candidate forreceiving a treatment by an ARHGAP26 function inhibitor and/or apharmaceutical agent for blocking an abnormal signal induced by a fusiongene composed of an OCLN gene and an ARHGAP26 gene, comprising a stepaccording to any one of [1] to [21], wherein the subject is a cancerpatient.

[34] The method according to [33] comprising a step of obtaining asample from a subject.

[35] The method according to [33] or [34] further comprising a step inwhich it is judged when a fusion gene composed of an OCLN gene and anARHGAP26 gene is detected in a sample obtained from a subject, that thesubject is a candidate for receiving a treatment by an ARHGAP26inhibitor and/or a pharmaceutical agent for blocking an abnormal signalinduced by a fusion gene composed of an OCLN gene and an ARHGAP26 gene.

[36] The method according to any one of [33] or [35], wherein cancer isstomach cancer.

Further, the present invention relates to [37] to [42] shown below.

[37] A primer set for detecting a fusion gene composed of an OCLN geneand an ARHGAP26 gene existing in a sample obtained from a subject, theprimer set comprising a sense primer designed from a section encodingOCLN and an antisense primer designed from a section encoding ARHGAP26,wherein the antisense primer consists of an oligonucleotide thathybridizes under a stringent condition with the polynucleotide accordingto any one of [1] to [5], and the sense primer consists of anoligonucleotide that hybridizes under a stringent condition with acomplementary strand of the polynucleotide.

[38] The primer set according to [37], wherein the sense primer consistsof an oligonucleotide that hybridizes under a stringent condition with acomplementary strand of a polynucleotide consisting of base no. 1 to 891of SEQ ID NO: 1, and the antisense primer consists of an oligonucleotidethat hybridizes under a stringent condition with a polynucleotideconsisting of base no. 892 to 2064 of SEQ ID NO: 1.

[39] A primer set for detecting a fusion gene composed of an OCLN geneand an ARHGAP26 gene existing in a sample obtained from a subject, theprimer set comprising a sense primer designed from a section encodingOCLN or an antisense primer designed from a section encoding ARHGAP26 ofthe polynucleotide according to any one of [1] to [5].

[40] The primer set according to any one of [37] to [39], wherein thesense primer consists of an oligonucleotide of at least 16 randomconsecutive bases between base no. 1 to 891 of SEQ ID NO: 1, and theantisense primer consists of an oligonucleotide that is complementary toan oligonucleotide of at least 16 random consecutive bases between baseno. 892 to 2064 of SEQ ID NO: 1.

[41] The primer set according to any one of [37] to [40], wherein thesubject is a cancer patient.

[42] The primer set according to [41], wherein cancer is stomach cancer.

Further, the present invention relates to [43] to [48] shown below.

[43] A probe for detecting a fusion gene composed of an OCLN gene and anARHGAP26 gene existing in a sample obtained from a subject, the probecomprising an oligonucleotide that hybridizes under a stringentcondition with the polynucleotide according to any one of [1] to [5].

[44] The probe set comprising multiple probes according to [43], theprobe set comprising a probe designed from a section encoding OCLN and aprobe designed from a section encoding ARHGAP26 of the polynucleotideaccording to any one of [1] to [5].

[45] The probe set according to [44] comprising multiple types of probesdesigned from a section encoding OCLN and multiple types of probesdesigned from a section encoding ARHGAP26.

[46] The probe set according to [44] or [45] comprising multiple typesof adjacent probe pairs comprising an oligonucleotide that iscomplementary to an oligonucleotide of at least 16 random consecutivebases between base no. 1 to 891 of SEQ ID NO: 1 and multiple types ofadjacent probe pairs comprising an oligonucleotide that is complementaryto an oligonucleotide of at least 16 random consecutive bases betweenbase no. 892 to 2064 of SEQ ID NO: 1.

[47] The probe or a probe set according to any one of [43] to [46],wherein the subject is a cancer patient.

[48] The probe or the probe set according to [47], wherein cancer isstomach cancer.

Further, the present invention relates to [49] to [53] shown below.

[49] A detection kit for detecting a fusion gene composed of an OCLNgene and an ARHGAP26 gene existing in a sample obtained from thesubject, the detection set comprising a primer set according to any oneof [37] to [40].

[50] A detection kit for detecting a fusion gene composed of an OCLNgene and an ARHGAP26 gene existing in a sample obtained from a subject,the detection kit comprising a probe or a probe set according to any oneof [43] to [46].

[51] The detection kit according to [50] further comprising a reagentfor amplifying a signal of hybridization.

[52] The detection kit according to any one of [49] to [51], wherein thesubject is a cancer patient.

[53] The detection kit according to [52], wherein cancer is stomachcancer.

Further, the present invention relates to [54] to [63] shown below.

[54] A detection method of a fusion protein of OCLN and ARHGAP26comprising a step of detecting whether a polypeptide according to either(1) or (2) exists in a sample obtained from a subject:

(1) a polypeptide that comprises an amino acid sequence having no lessthan 90% identity with an amino acid sequence represented by SEQ ID NO:2;

(2) a polypeptide that comprises an amino acid sequence represented bySEQ ID NO: 2, or a polypeptide that comprises an amino acid sequencerepresented by SEQ ID NO: 2, in which 1 to 10 amino acids are deleted,substituted, inserted and/or added.

[55] The method according to [54], wherein the polypeptide comprises anamino acid sequence having no less than 90% identity with an amino acidsequence represented by SEQ ID NO: 2, and has an ability to developtumor.

[56] The method according to [54], wherein the polypeptide comprises anamino acid sequence represented by SEQ ID NO: 2 and has an ability todevelop tumor, or the polypeptide comprises an amino acid sequencerepresented by SEQ ID NO: 2, in which 1 to 10 amino acids are deleted,substituted, inserted and/or added, and has an ability to develop tumor.

[57] The method according to [54], wherein the polypeptide consists ofan amino acid sequence represented by SEQ ID NO: 2.

[58] A detection method of a fusion protein of OCLN and ARHGAP26comprising a step of detecting whether a polypeptide consisting of anamino acid sequence represented by SEQ ID NO: 2 exists in a sampleobtained from a subject.

[59] The method according to any one of [54] to [58], wherein the stepfor detecting whether the polypeptide exists comprises a step ofbringing an antibody (primary antibody) that recognizes a sectionderived from an OCLN gene in the polypeptide and an antibody (primaryantibody) that recognizes a section derived from an ARHGAP26 gene in thepolypeptide in contact with a sample obtained from a subject.

[60] The method according to [59] further comprising steps of i) to v)described below:

i) a step of adding secondary antibodies that are connected tooligonucleotides and that respectively bind to primary antibodies; ii) astep of adding a ligation solution that contains two types ofoligonucleotides that are partially complementary to oligonucleotidesconnected to the secondary antibodies and a ligase that can ligate thetwo types of oligonucleotides to form a circular structure when theoligonucleotides approach each other, thereby inducing a ligationreaction; iii) a step of elongating a nucleic acid along a circularstructure that is formed; and iv) a step of hybridizing a labeledoligonucleotide probe that can hybridize with an elongated nucleic acid,and v) a step of detecting a labeled signal.

[61] The method according to any one of [54] to [60] comprising a stepof obtaining a sample from a subject.

[62] The method according to any one of [54] to [61], wherein thesubject is a cancer patient.

[63] The method according to [62], wherein cancer is stomach cancer.

Further, the present invention relates to [64] to [66] shown below.

[64] A method for detecting whether cancer exists in a subjectcomprising the step according to any one of [54] to [60].

[65] The method according to [64] comprising a step of obtaining asample from a subject.

[66] The method according to [64] or [65], wherein cancer is stomachcancer.

Further, the present invention relates to [67] to [71] shown below.

[67] A method for diagnosing cancer in a subject comprising a stepaccording to any one of [54] to [60].

[68] The method according to [67] comprising a step of obtaining asample from a subject.

[69] The method according to [67] or [68] further comprising a step inwhich it is judged when a fusion protein of OCLN and ARHGAP26 isdetected in a sample obtained from a subject, that there is a highpossibility of the subject having cancer.

[70] The method according to [67] or [68], wherein cancer is stomachcancer.

[71] The method according to [68] further comprising a step in which itis judged when a fusion protein of OCLN and ARHGAP26 is detected in asample obtained from a subject, that there is a high possibility of thesubject having stomach cancer.

Further, the present invention relates to [72] to [75] shown below.

[72] A method for identifying a subject that is a candidate forreceiving a treatment by an ARHGAP26 function inhibitor and/or apharmaceutical agent for blocking an abnormal signal induced by a fusiongene composed of an OCLN gene and an ARHGAP26 gene, the methodcomprising a step according to any one of [54] to [60], wherein thesubject is a cancer patient.

[73] The method according to [72] comprising a step of obtaining asample from a subject.

[74] The method according to [72] or [73] further comprising a step inwhich it is judged when a fusion protein of OCLN and ARHGAP26 isdetected in a sample obtained from a subject, that the subject is acandidate for receiving a treatment by an ARHGAP26 inhibitor and/or apharmaceutical agent for blocking an abnormal signal induced by a fusiongene composed of an OCLN gene and an ARHGAP26 gene.

[75] The method according to [72] or [74], wherein cancer is stomachcancer.

Further, the present invention relates to [76] to [79] shown below.

[76] A detection kit for detecting a fusion protein of OCLN and ARHGAP26existing in a sample obtained from a subject, the detection kitcomprising an antibody (primary antibody) that recognizes a sectionderived from an OCLN gene in the polypeptide according to any one of[54] to [58], and an antibody (primary antibody) that recognizes asection derived from an ARHGAP26 gene in said polypeptide.

[77] The detection kit according to [76] comprising secondary antibodiesthat are connected to oligonucleotides and that respectively bind toprimary antibodies, two types of oligonucleotides that are partiallycomplementary to the oligonucleotides connected to the secondaryantibodies, a ligase that can ligate the two types of oligonucleotidesto form a circular structure when the oligonucleotides approach eachother, and a labeled oligonucleotide probe.

[78] The detection kit according to [76] or [77], wherein the subject isa cancer patient.

[79] The detection kit according to [78], wherein cancer is stomachcancer.

Further, the present invention relates to [80] to [81] shown below.

[80] A polypeptide according to any one of (1) to (3) shown below or apolynucleotide encoding said polypeptide:

(1) a polypeptide that comprises an amino acid sequence having no lessthan 90% identity with an amino acid sequence represented by SEQ ID NO:2;

(2) a polypeptide that comprises an amino acid sequence represented bySEQ ID NO: 2, in which 1 to 10 amino acids are deleted, substituted,inserted and/or added;

(3) a polypeptide consisting of an amino acid sequence represented bySEQ ID NO: 2.

[81] The polypeptide or a polynucleotide encoding said polypeptideaccording to [80] that has an ability to develop tumor.

Advantageous Effect of Invention

The detection method of the present invention may be used as a methodfor detecting cancer (particularly, stomach cancer) that tests positivefor a fusion gene composed of an OCLN gene and an ARHGAP26 gene(hereinafter referred to as OCLN-ARHGAP26 fusion gene). The primer set,probe, probe set and detection kit of the present invention may be usedin a detection method of the present invention.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the result of the Western blot. It shows the change in theamount of protein expression of the OCLN-ARHGAP26 fusion protein by theARHGAP26 siRNA treatment.

FIG. 2 shows the change in the number of viable cells in the stomachcancer cell line caused by the ARHGAP26 siRNA treatment. FIG. 2 comparesthe number of viable cells cultured in a 0.5% bovine serum containingRPMI-1640 medium after introduction of siRNA with that of the control.

FIG. 3 shows a result of amplification by PCR of a region containing afusion point of an OCLN-ARHGAP26 fusion gene.

FIG. 4 shows a change caused by an OCLN siRNA treatment, and an ARHGAP26siRNA treatment in the number of viable cells in the stomach cancer cellline over time.

DESCRIPTION OF EMBODIMENTS

Detection Method of the Present Invention

The detection method of the present invention includes a method fordetecting a fusion gene, and a method for detecting a fusion proteinencoded in the fusion gene. The method for detecting a fusion gene ofthe present invention or the method for detecting a fusion protein ofthe present invention includes a step of detecting whether a specificpolynucleotide or polypeptide exists in a sample obtained from asubject.

Items collected from the subject (samples separated from a living body)are used as the sample obtained from the subject, specifically, anycells, tissues, or body fluids that were collected (blood, oral mucus,circulating tumor cells, exosome, etc.), biopsied samples (samples fromthe primary focus, cancer cells in the peritoneal lavage solution,cancer cells in ascites, etc.), of which the biopsied samples arepreferred. It is possible to use genome DNAs extracted from thecollected samples or to use transcription products thereof (productsthat are obtained by transcription and translation of a genome; e.g.RNA, protein) or cDNA prepared from RNA. Using RNA or cDNA that had beenformulated is preferred. It is also possible to use a stabilized samplefixed in formalin and embedded in paraffin (Formalin-FixedParaffin-Embedded sample; FFPE Sample). A FFPE sample sliced into a thinFFPE slice may also be used. A use of a FFPE slice enables a directdetection of a polynucleotide existing in the slice.

The method for detecting a fusion gene in the present invention is amethod for detecting “a fusion gene composed of an OCLN gene and anARHGAP26 gene,” wherein the fusion gene is a fusion gene comprising apart of an OCLN gene and a part of an ARHGAP26 gene. An exemplary fusiongene composed of an OCLN gene and an ARHGAP26 gene includes apolynucleotide consisting of a base sequence represented by SEQ IDNO: 1. The polynucleotide consisting of a base sequence represented bySEQ ID NO: 1 is a polynucleotide with a base sequence of base no. 207(corresponding to the 5′ terminal of the coding sequence (hereinafterreferred to as CDS)) to 1097 of an OCLN gene (GenBank registration no:NM_001205254.1) and base no. 1143 to 2315 (corresponding to the 3′terminal of CDS) of an ARHGAP26 gene (GenBank registration no:NM_001135608.1), in which thymine at base no. 1280 is substituted withguanine, and cytosine at base no. 2225 is substituted with thymine. Ofthe base sequence represented by SEQ ID NO: 1, the sequence from baseno. 1 to 891 is derived from an OCLN gene, and the sequence from baseno. 892 to 2064 is derived from an ARHGAP26 gene. The polynucleotideconsisting of a base sequence represented by SEQ ID NO: 1 is alsoreferred to as a “fusion polynucleotide.” The amino acid sequenceencoded in base no. 1 to 2064 of SEQ ID NO: 1 is shown in SEQ ID NO: 2.

In the “step of detecting whether a polynucleotide exists” in thedetection method of a fusion gene of the present invention, thepolynucleotide that is the target of detection (referred to in thepresent specification as the “polynucleotide targeted in detection”)includes, for example, a polynucleotide encoding a polypeptide describedin (1) or (2) shown below:

(1) a polypeptide that comprises an amino acid sequence having no lessthan 90% identity with an amino acid sequence represented by SEQ ID NO:2;(2) a polypeptide that comprises an amino acid sequence having no lessthan 90% identity with an amino acid sequence represented by SEQ ID NO:2, and has an ability to develop tumor.

In the aforementioned polypeptide, the “identity with an amino acidsequence represented by SEQ ID NO: 2” is preferably 95% or higher, andmore preferably 98% or higher.

Note that the “identity” as used in the present specification is a valueof “Identity” obtained by using a parameter prepared by default by theNEEDLE program (J Mol Biol 1970; 48: 443-453) search. The aforementionedparameter is shown below.

Gap penalty=10Extend penalty=0.5

Matrix=EBLOSUM62

Whether a polypeptide “has an ability to develop tumor” or not may beconfirmed by a method shown below in Example 2. One specific method isto introduce siRNA that suppresses the expression of a polynucleotideencoding the polypeptide to a cell expressing the polypeptide (stomachcancer cell line OKAJIMA), and to verify that the viability of the celldecreases.

In one embodiment of the present invention, the polynucleotide targetedin detection is a polynucleotide encoding a polypeptide according to anyone of (1) to (4) shown below:

(1) a polypeptide that comprises an amino acid sequence represented bySEQ ID NO: 2, in which 1 to 10 amino acids are deleted, substituted,inserted and/or added;

(2) a polypeptide that comprises an amino acid sequence represented bySEQ ID NO: 2, in which 1 to 10 amino acids are deleted, substituted,inserted and/or added, and has an ability to develop tumor;

(3) a polypeptide that comprises an amino acid sequence represented bySEQ ID NO: 2 and has an ability to develop tumor; and

(4) a polypeptide that consists of an amino acid sequence represented bySEQ ID NO: 2.

In the polypeptide of (1) and (2), the number of amino acids that hadbeen deleted, substituted, inserted and/or added in the amino acidsequence represented by SEQ ID NO: 2 is preferably one to a few, morepreferably 1 to 7, and even more preferably 1 to 5.

An example of a polynucleotide that encodes “a polypeptide that consistsof an amino acid sequence represented by SEQ ID NO: 2” includes “apolynucleotide that consists of a base sequence represented by SEQ IDNO: 1.”

The method for detecting a fusion gene of the present invention maycomprise a step in which it is judged whether the polynucleotidetargeted in detection exists by whether the polynucleotide was detected.

The method for detecting a fusion gene of the present invention mayfurther comprise a step in which it is judged when a polynucleotidetargeted in detection is detected, that a fusion gene composed of anOCLN gene and an ARHGAP26 gene exists.

The method for detecting a fusion gene of the present invention maycomprise a step of amplifying the nucleic acid existing in the sampleobtained from a subject or a step of hybridizing a probe with thenucleic acid existing in the sample obtained from a subject to detectthe polynucleotide targeted in detection.

The nucleic acid for use may be a genome DNA, RNA or a cDNA preparedfrom RNA. The methods of extracting DNA, extracting RNA or preparingcDNA from RNA is commonly known in the field, and it may be performedeasily by using a commercially available DNA extraction kit, RNAextraction kit or a cDNA synthesis kit.

The step of amplifying a nucleic acid in the sample obtained from asubject may be performed by a commonly known method of amplifying anucleic acid. Such method includes PCR (Polymerase chain reaction, e.g.real-time PCR), LCR (Ligase chain reaction), SDA (Strand displacementamplification), NASBA (Nucleic acid sequence-based amplification), ICAN(Isothermal and chimeric primer-initiated amplification of nucleicacids), LAMP (Loop-mediated isothermal amplification), TMA(Transcription-mediated amplification, e.g. Gen-Probe's TMA system), anda preferable method is PCR.

Specifically, the nucleic acid (e.g. genome DNA, RNA, or cDNA preparedfrom RNA, etc.) in the sample obtained from a subject is subjected to anucleic acid amplification reaction using a primer set designed tospecifically amplify a polynucleotide targeted in detection. The primerset to be used is not particularly limited as long as it canspecifically amplify a polynucleotide targeted in detection. Forexample, a use of a primer design software (e.g. Primer Express; AppliedBiosystems) allows a person skilled in the art to easily design theprimer set based on the base sequence of a polynucleotide targeted indetection. More specifically, a primer set includes a sense primer(5′-primer) designed from a section that encodes the OCLN of apolynucleotide targeted in detection (e.g. any section in an OCLN generegion of the fusion polynucleotide (particularly, cDNA)) and anantisense primer (3′-primer) designed from a section encoding ARHGAP26of a polynucleotide targeted in detection (e.g. any section in anARHGAP26 gene region of the fusion polynucleotide (particularly, cDNA)),and the antisense primer consists of an oligonucleotide that hybridizeswith a polynucleotide targeted in detection under a stringent condition(preferably, under a highly stringent condition), and the sense primerconsists of an oligonucleotide that hybridizes with a complementarystrand of a polynucleotide targeted in detection under a stringentcondition (preferably, under a highly stringent condition). Otherwise,either the sense primer or the antisense primer may be designed so thatit corresponds to the region comprising the fusion point of thepolynucleotide targeted in detection.

The “stringent condition” in the present specification refers to ahybridization condition of “5×SSPE, 5×Denhardt's solution, 0.5% SDS, 50%formaldehyde, 200 μg/mL salmon sperm DNA, at 42° C. overnight” and awashing condition of “0.5×SSC, 0.1% SDS, 42° C.” “A highly stringentcondition” refers to a hybridization condition of “5×SSPE, 5×Denhardt'ssolution, 0.5% SDS, 50% formaldehyde, 200 μg/mL salmon sperm DNA, at 42°C. overnight” and a washing condition of “0.2×SSC, 0.1% SDS, 65° C.”

The “fusion point” of the polynucleotide targeted in detection in thepresent specification is a point in which a section derived from an OCLNgene and a section derived from an ARHGAP26 gene in the polynucleotidetargeted in detection are fused together, and the “region comprising thefusion point” in the polynucleotide targeted in detection is, forexample, the region comprising bases of base no. 891 and 892 when thepolynucleotide targeted in detection is a polynucleotide consisting of abase sequence represented by SEQ ID NO: 1.

In an embodiment of the present invention, the sense primer consists ofan oligonucleotide hybridizing with a complementary strand of apolynucleotide that consists of base no. 1 to 891 of SEQ ID NO: 1 undera stringent condition, and the antisense primer consists of anoligonucleotide hybridizing with a polynucleotide that consists of baseno. 892 to 2064 of SEQ ID NO: 1 under a stringent condition.

In an embodiment of the present invention, the sense primer consists ofat least 16 consecutive bases of an oligonucleotide between base no. 1to 891 of SEQ ID NO: 1, and the antisense primer consists of anoligonucleotide that is complementary with at least 16 consecutive basesof an oligonucleotide that consists of base no. 892 to 2064 of SEQ IDNO: 1.

In a step to amplify nucleic acid, the sense primer and the antisenseprimer should preferably be set so that the fragment size of the nucleicacid to be amplified is 1 kb or lower, since a large fragment size ofthe nucleic acid to be amplified leads to poor amplification efficiency.The primers to be used generally have a chain length of at least 15bases, preferably at least 16 bases, more preferably at least 18 bases,even more preferably at least 20 bases. In one embodiment of the presentinvention, the primer has 15 to 40 bases, preferably 16 to 24 bases,more preferably 18 to 24 bases, even more preferably 20 to 24 bases.

The primer may be produced by chemical synthesis without beingparticularly limited thereby.

In a preferable embodiment, the detection method of a fusion gene of thepresent invention further encompasses a step of detecting whether anamplified nucleic acid fragment of a desired size was obtained inaddition to a step of amplifying nucleic acid in the sample obtainedfrom a subject. The step of detecting whether an amplified nucleic acidfragment of a desired size was obtained may be performed usingelectrophoresis. By using electrophoresis, the nucleic acid fragment maybe analyzed by agarose gel electrophoresis to confirm whether amplifiednucleic acid fragments were produced in the desired size by usingethidium bromide dye, etc.

Further, by performing a PCR amplification monitor in the amplificationprocess of the gene (real time PCR) (Genome Res. 1996; 6(10): 986-994),it is possible to perform a quantified analysis of amplified nucleicacid fragments. A possible candidate to be used in the PCR amplificationmonitoring method is ABI PRISM7900 (Applied Biosystems).

When an amplified nucleic acid fragment of the desired size is obtained,that means that a polynucleotide targeted in detection existed in thesample obtained from a subject. The detection method of a fusion gene ofthe present invention may further include a step in which it is judgedwhen an amplified nucleic acid fragment of the desired size is obtained,that a fusion gene composed of an OCLN gene and an ARHGAP26 gene exists.

In a separate preferable embodiment, the detection method of the fusiongene of the present invention further encompasses a step of determiningthe base sequence of the amplified nucleic acid in addition to a step ofamplifying the nucleic acid of the sample obtained from a subject. Thestep of determining the base sequence of the nucleic acid fragment mayuse sequencing methods commonly known in the field of art including nextgeneration sequencing methods (Nature Biotechnology 2008; 26: 1135-1145)(e.g. HiSeq2500 (Illumina)), such as the Sanger sequencing (e.g. ABIPRISM3100 (Applied Biosystems) may be used), or sequencing by synthesis,etc.

The step of determining the base sequence of the nucleic acid fragmentincludes not just a step of sequencing the full length of a nucleic acidfragment, but a step of sequencing partial sequences corresponding toboth ends of the nucleic acid fragment.

When the sequenced nucleic acid fragment includes a base sequence of asection encoding OCLN and a base sequence of a section encoding ARHGAP26of the polynucleotide targeted in detection in the same fragment, thatmeans that the polynucleotide targeted in detection existed in thesample obtained from a subject. The detection method of the fusion geneof the present invention may further include a step in which it isjudged when the amplified nucleic acid fragment includes a base sequenceof a section encoding OCLN and a base sequence of a section encodingARHGAP26 of the polynucleotide targeted in detection in the samefragment, that a fusion gene composed of an OCLN gene and an ARHGAP26gene exists.

The step of hybridizing a probe with a nucleic acid in the sampleobtained from a subject may be performed using a probe includingoligonucleotide that hybridizes under a stringent condition (preferably,under a highly stringent condition) with a polynucleotide targeted indetection, and using a commonly known hybridization method. Such methodsinclude, for example, Northern hybridization, dot blot method, DNA microarray method, RNA protection method, in situ hybridization, etc. Apreferable method is the in situ hybridization. Detection using the insitu hybridization may be performed by a commonly known fluorescent insitu hybridization (FISH), chromogenic in situ hybridization (CISH), orsilver in situ hybridization (SISH). The chain length of the probe usedin hybridization may be selected as necessary by a person skilled in theart according to the hybridization method to be used, but the probepreferably has a chain length of at least 16 bases.

In one embodiment of the present invention, the probe used inhybridization is an oligonucleotide that hybridizes under a stringentcondition (preferably, under a highly stringent condition) with apolynucleotide targeted in detection, or a complementary strand thereof,and it includes an oligonucleotide of at least 16 bases upstream and atleast 16 bases downstream of the fusion point on the polynucleotidetargeted in detection (a specific example being a sequence of base no.876 to 907 in SEQ ID NO: 1) or an oligonucleotide that is complementaryto said oligonucleotide.

In one embodiment of the present invention, the step of hybridizing aprobe with a nucleic acid existing in a sample obtained from a subjectmay be performed according to the commonly known RNA FISH method (J.Mol. Diagn. 2012; 14(1): 22-29). More specifically, in situhybridization is performed using a sample obtained from a subject (e.g.FFPE fragment), a probe designed from a section encoding OCLN of thepolynucleotide targeted in detection (e.g. any section in an OCLN generegion of the fusion polynucleotide), and a probe designed from asection encoding ARHGAP26 of the polynucleotide targeted in detection(e.g. any section in an ARHGAP26 gene region of the fusionpolynucleotide). The probes include oligonucleotides that hybridizeunder a stringent condition (preferably, under a highly stringentcondition) with the polynucleotide targeted in detection.

In one embodiment of the present invention, the in situ hybridization isperformed using multiple detection probes designed from a sectionencoding OCLN and multiple detection probes designed from a sectionencoding ARHGAP26.

In one embodiment of the present invention, the in situ hybridization isperformed using the following probes:

multiple types of adjacent probe pairs including oligonucleotides thatare complementary to at least 16 random consecutive oligonucleotides inbase no. 1 to 891 of SEQ ID NO: 1 (preferably 10 to 25 types, morepreferably 18 to 22 types, even more preferably 20 types of probepairs), and multiple types of adjacent probe pairs includingoligonucleotides that are complementary to at least 16 randomconsecutive oligonucleotides in base no. 892 to 2064 of SEQ ID NO: 1(preferably 10 to 25 types, more preferably 18 to 22 types, even morepreferably 20 types of probe pairs).

The “adjacent probe pairs” in the present specification consist of twotypes of probes that are arranged next to each other when they hybridizewith the polynucleotide targeted in detection. The probes include anoligonucleotide that is complementary to the polynucleotide targeted indetection, and the length of the oligonucleotide is generally at least16 bases, preferably at least 18 bases. In one embodiment of the presentinvention, the length of the oligonucleotide is 16 to 30 bases,preferably 18 to 25 bases.

In a preferable embodiment of the present invention, the detectionmethod of the fusion gene of the present invention further encompasses astep of amplifying a hybridization signal in addition to a step ofperforming in situ hybridization. To perform a step of amplifying ahybridization signal, a reagent that amplifies a hybridization signalmay be hybridized with a probe that hybridizes with a nucleic acidcontained in the sample.

Reagents that amplify a hybridization signal used in in situhybridization include PreAmplifier Mix QT, Amplifier Mix QT, Label ProbeMix, and Label Probe Diluent QF, which may be obtained from Affymetrix.

In a more preferable embodiment, the detection method of the fusion geneof the present invention further encompasses a step of detecting asignal overlap between a signal from a probe designed from a sectionencoding OCLN and a signal from a probe designed from a section encodingARHGAP26. By separating the fluorescent reagent or the color reagentthat detects a probe designed from a section encoding OCLN and a probedesigned from a section encoding ARHGAP26, it is possible to observewhether the signals from the two different probes are in the same area(inside the same molecule). When it is observed that the signals fromthe two different probes are in the same area (inside the samemolecule), that would mean that the polynucleotide targeted in detectionexisted in the sample obtained from a subject. The detection method ofthe fusion gene of the present invention may further include a step inwhich it is judged when the two signals are in the same area (inside thesame molecule), that a fusion gene composed of an OCLN gene and anARHGAP26 gene exists.

The probes are not particularly limited, but they may be produced by achemical synthesis method.

The detection method of the fusion protein of the present invention is amethod for detecting “a fusion protein of OCLN and ARHGAP26” and thefusion protein is a fusion protein encoded by the fusion gene of theOCLN gene and the ARHGAP26 gene.

In the “step of detecting whether polypeptide exists” in the detectionmethod of the fusion protein of the present invention, the polypeptidetargeted in detection includes a polypeptide that is encoded by apolynucleotide targeted in detection.

The detection method of the fusion protein of the present invention mayencompass a step in which it is judged whether a polynucleotide existsby whether the polypeptide targeted in detection is detected.

The detection method of the fusion protein of the present invention mayfurther encompass a step in which it is judged when the polypeptidetargeted in detection is detected, that a fusion protein of OCLN andARHGAP26 exists.

The step of detecting whether a polypeptide exists may be performed bypreparing a lysate derived from a sample obtained from a subject (e.g.cancer tissue or cell obtained from a subject) and measuring thepolypeptide targeted in detection, contained in the sample by animmunological measurement method or an enzyme active measurement method,which combine antibodies against proteins that constitute the fusionprotein, or a detection method that combines these methods, or by massspectrometry. Further, this step may be performed by a detection methodusing an immunological tissue staining technology performed by combiningthe polypeptide targeted in detection included in the sample (e.g. FFPEfragment) obtained from a subject, that had appropriately undergonepretreatment (such as, removal of paraffin), with the antibodies againstproteins constituting the fusion protein. Otherwise, this step may beperformed by exchanging the antibodies against proteins constituting thefusion protein to antibodies that recognize the fusion section of thefusion protein. Exemplary approaches to these methods include thefollowing methods using monoclonal antibodies and polyclonal antibodiesspecific to the polypeptide targeted in detection: enzyme immunizingmeasurement, double antibody sandwich ELISA method, fluorescentimmunological measurement method, radioimmunological measurement method,Western blot, immunohistologic staining, a detection method combiningimmune precipitation and mass spectrometry, etc.

The “fusion section” of the fusion protein of the present specificationrefers to a section in the polypeptide targeted in detection, in whichthe section derived from an OCLN gene and a section derived from anARHGAP26 gene are fused.

The detection using an immunohistologic staining technology may beperformed according to Proximity Ligation Assay (Nat. Methods. 2006;3(12): 995-1000). More specifically, whether the polypeptide targeted indetection exists or not may be detected by using an antibody thatrecognizes a section derived from the OCLN gene of the polypeptidetargeted in detection, and an antibody that recognizes a section derivedfrom an ARHGAP26 gene of a polypeptide targeted in detection, and bydetecting that the two antibodies recognize the same molecule by theaforementioned technologies. More specifically, the detection may beperformed by i) a step of bringing an antibody (primary antibody) thatrecognizes a section derived from an OCLN gene of polypeptide targetedin detection, and the antibody (primary antibody) that recognizes asection derived from an ARHGAP26 gene of polypeptide targeted indetection, in contact with the sample obtained from the subject; ii) astep of adding secondary antibodies that are connected tooligonucleotides, and binds to the respective primary antibodies, iii) astep of inducing ligation by adding two types of oligonucleotides thatare partly complementary to oligonucleotides connected to the secondaryantibodies, and a ligation solution containing ligase that can form acircular structure by ligation of the two types of oligonucleotides whenthey approach each other; iv) a step of elongating a nucleic acid alongthe circular structure that was formed, v) a step of hybridizing alabeled oligonucleotide probe that can hybridize with the elongatednucleic acid; and vi) a step of detecting the labeling signal. Suchdetection may be performed using a PLA probe and reagents included inthe Duolink II reagent kit or the Duolink II Bright field reagent kit(Olink).

In one embodiment of the present invention, the detection method of thepresent invention encompasses a step of obtaining a sample from thesubject.

In one embodiment of the present invention, the subject of the detectionmethod of the present invention is a cancer patient, and in a morespecific embodiment, the cancer is stomach cancer. The type of stomachcancer is not particularly limited, but it may be a diffuse type, anintestinal type, or a mix type in the Lauren classification. Further,without being limited thereby, the stomach cancer may be any ofpapillary adenocarcinoma, tubular adenocarcinoma, poorly differentiatedadenocarcinoma, signet ring cell carcinoma, or carcinoma mucoid, etc.

In the detection method of the present invention, it is possible tojudge when the polynucleotide targeted in detection, or the polypeptidetargeted in detection is detected in the sample obtained from thesubject, that the subject has cancer (particularly, stomach cancer).

The detection step in the detection method of the present invention maybe used as a method for detecting whether cancer (particularly, stomachcancer) exists in a subject or a method for diagnosing cancer(particularly, stomach cancer) in the subject. The diagnosis method ofthe present invention may include, in addition to the aforementioneddetection step, a step in which it is judged when the polynucleotidetargeted in detection, or the polypeptide targeted in detection isdetected in the sample obtained from the subject, that there is a highpossibility that the subject has cancer (particularly, stomach cancer).Further, the detection step may be used in a method for identifying asubject (a cancer patient of stomach cancer, etc.) that is a candidatefor receiving a treatment by an ARHGAP26 function inhibitor and/or apharmaceutical agent that blocks abnormality signal induced by a fusiongene composed of an OCLN gene and an ARHGAP26 gene. The identificationmethod of the present invention may include, in addition to thedetection step, a step in which it is judged when a polynucleotide isdetected in a sample obtained from the subject, that the subject is acandidate for receiving a treatment by an ARHGAP26 function inhibitorand/or a pharmaceutical agent that blocks abnormality signal induced bya fusion gene composed of an OCLN gene and an ARHGAP26 gene.

The Primer Set, Probe, Probe Set and Detection Kit of the PresentInvention

The present invention encompasses a primer set, probe, probe set and adetection kit used in the detection method of the present invention.

The primer set of the present invention includes a sense primer designedfrom a section encoding OCLN and an antisense primer designed from asection encoding ARHGAP26, and the antisense primer consists of anoligonucleotide that hybridizes with the polynucleotide targeted indetection under a stringent condition (preferably, under a highlystringent condition), and the sense primer consists of anoligonucleotide that hybridizes with a complementary strand of apolynucleotide targeted in detection under a stringent condition(preferably, under a highly stringent condition).

In the primer set of the present invention, either the sense primer orthe antisense primer may be designed so that it corresponds to a regionin a polynucleotide targeted in detection that comprises a fusion point.

A specific embodiment of the primer set of the present inventionincludes the following primer set:

a primer set consisting of a sense primer consisting of anoligonucleotide that hybridizes under a stringent condition with acomplementary strand of a polynucleotide consisting of base no. 1 to 891of SEQ ID NO: 1 and an antisense primer consisting of an oligonucleotidethat hybridizes under a stringent condition with a polynucleotideconsisting of base no. 892 to 2064 of SEQ ID NO: 1.

A more specific embodiment of the primer set of the present inventionincludes the following primer set:

a primer set consisting of a sense primer consisting of anoligonucleotide of at least 16 random consecutive bases between base no.1 to 891 of SEQ ID NO: 1 and an antisense primer consisting of anoligonucleotide that is complementary with at least 16 randomconsecutive bases between base no. 892 to 2064 of SEQ ID NO: 1.

It is preferable for the primer set to have a space of 1 kb or lowerbetween the selected positions of the sense primer and the antisenseprimer, or a nucleic acid fragment amplified by the sense primer and theantisense primer with a size of 1 kb or lower. Further, the primer ofthe present invention normally has a chain length of at least 15 bases,preferably at least 16 bases, more preferably at least 18 bases, evenmore preferably at least 20 bases. In one embodiment of the presentinvention, the primer has a chain length of 15 to 40 bases, preferably16 to 24 bases, more preferably 18 to 24 bases, and even more preferably20 to 24 bases.

The primers included in the primer set of the present invention, withoutbeing particularly limited, may be produced by a chemical synthesismethod.

The probes included in the probe of the present invention and the probeset of the present invention includes an oligonucleotide that hybridizeswith the polynucleotide targeted in detection under a stringentcondition (preferably, under a highly stringent condition). The chainlength of the probes included in the probe of the present invention orthe probe set of the present invention may be selected as necessary by aperson skilled in the art according to the applied hybridization method,but the probe preferably has a chain length of at least 16 bases.

In one embodiment of the present invention, the probe of the presentinvention includes an oligonucleotide of at least 16 bases upstream andat least 16 bases downstream of the fusion point in the polynucleotidetargeted in detection (specifically, the sequence between base no. 876to 907 of SEQ ID NO: 1), or an oligonucleotide that is complementarythereto.

In one embodiment of the present invention, the probe set of the presentinvention includes a probe designed from a section encoding OCLN (e.g.any section in the OCLN gene region of the fusion polynucleotide) and aprobe designed from a section encoding ARHGAP26 (e.g. any section in theARHGAP26 gene region of the fusion polynucleotide).

In one embodiment of the present invention, the probe set of the presentinvention includes multiple types of probes designed from a sectionencoding OCLN and multiple types of probes designed from a sectionencoding ARHGAP26.

In one embodiment of the present invention, the probe set of the presentinvention includes the following:

multiple types of adjacent probe pairs including an oligonucleotide thatis complementary to an oligonucleotide of at least 16 random consecutivebases between base no. 1 to 891 of SEQ ID NO: 1 (preferably 10 to 25types, more preferably 18 to 22 types, even more preferably 20 types ofprobe pairs), and multiple types of adjacent probe pairs including anoligonucleotide that is complementary with an oligonucleotide of atleast 16 random consecutive bases between base no. 892 to 2064 of SEQ IDNO: 1 (preferably 10 to 25 types, more preferably 18 to 22 types, evenmore preferably 20 types of probe pairs).

The probes of the probe pair include an oligonucleotide that iscomplementary with the polynucleotide targeted in detection, and thelength of the oligonucleotide is normally at least 16 bases, preferablyat least 18 bases. In one embodiment of the present invention, thelength of the oligonucleotide is 16 to 30 bases, preferably 18 to 25bases.

The probe of the present invention and the probe included in the probeset of the present invention, without being limited thereby, may beproduced by chemical synthesis.

The present invention encompasses a detection kit including a primer setof the present invention, a probe of the present invention or the probeset of the present invention. The detection kit of the present inventionmay include in addition to the primer set of the present invention, theprobe of the present invention or the probe set of the presentinvention, components that may be used together with the primer set, theprobe or the probe set for the detection of a polynucleotide targeted indetection such as reagents to amplify the signal of hybridization.

The present invention also encompasses a detection kit for detecting apolypeptide targeted in detection. Preferably, the detection kitincludes an antibody (primary antibody) that recognizes a sectionderived from an OCLN gene of polypeptide targeted in detection, and anantibody (primary antibody) that recognizes a section derived from anARHGAP26 gene of polypeptide targeted in detection. More preferably, thepresent invention may include secondary antibodies connected witholigonucleotides that are respectively bound to primary antibodies, twotypes of oligonucleotides that are partially complementary to theoligonucleotides connected to the secondary antibodies, ligase thatforms a circular structure by ligation of the two types ofoligonucleotides when they approach each other, and labeledoligonucleotide probes.

The primer set, probe, probe set, and detection kit of the presentinvention may be used for the detection method, diagnosis method,identification method of a patient, and identification method of asubject of the present invention. In one embodiment of the presentinvention, with respect to the primer set, probe, probe set anddetection kit of the present invention, the subject is a cancer patientand more specifically, the cancer is stomach cancer. The stomach canceris not particularly limited, but it may be a diffuse type, an intestinaltype, or a mix type in the Lauren classification. Further, without beinglimited thereby, the stomach cancer may be any of papillaryadenocarcinoma, tubular adenocarcinoma, poorly differentiatedadenocarcinoma, signet ring cell carcinoma, or carcinoma mucoid.

EXAMPLES

The Examples may be performed by commonly known methods unless otherwiseindicated. When using commercially available reagents or kits, theExamples may be performed according to the manuals of the commercialproducts.

Example 1 Isolation of OCLN-ARHGAP26 Fusion Gene

Total RNA was prepared from OKAJIMA, a stomach cancer cell line providedfrom First Department of Pathology, Hiroshima University School ofMedicine (currently, Department of Molecular Pathology, Graduate Schoolof Biomedical and Health Sciences, Hiroshima University), andreverse-transcribed into cDNA with a reverse transcriptase(SuperScriptIII; Life Technologies) and Oligo(dT) Primer (Oligo(dT)20Primer; Life Technologies) according to the standard protocol of thereagent.

Next, primers of OCLN_full fwd21 represented by SEQ ID NO: 3 andARHGAP26_full rev01 represented by SEQ ID NO: 4 were used to perform PCR(10 sec. at 98° C., 15 sec. at 55° C., and 3 min at 68° C., 30 cycles,followed by 5 min at 68° C.) using DNA polymerase (PrimeSTAR GXL; TAKARABIO INC.) with cDNA obtained in above step as a template. Then, usingthe aforementioned PCR product diluted by 10-fold as a template, primersof OCLN_full fwd22 represented by SEQ ID NO: 5 and ARHGAP26_full rev02represented by SEQ ID NO: 6 were used to perform PCR (10 sec. at 98° C.,15 sec. at 55° C., and 3 min at 68° C., 30 cycles, followed by 5 min at68° C.) using the same DNA polymerase. Electrophoresis was performedafter the PCR to obtain a PCR product of about 2 kbp. After adding A tothe 3′-end of the PCR product using Takara Taq (TAKARA BIO INC.), it wascloned into a cloning vector (TOPO XL PCR Cloning Kit; LifeTechnologies) and sequenced by dideoxy sequencing method (BigDyeTerminator v3.1 Cycle Sequencing Kit; Life Technologies). Consequently,the PCR product that is about 2 kbp derived from the stomach cancer cellline OKAJIMA was found to be a transcription product (SEQ ID NO: 1) inwhich a nucleotide sequence of base no. 207 (corresponding to the 5′terminal of CDS) to 1097 of OCLN (NM_001205254.1) registered in NCBI isfused to a nucleotide sequence of base no. 1143 to 2315 of ARHGAP26(NM_001135608.1) (corresponding to the 3′ terminal of CDS) withsubstitutions of thymine to guanine at base no. 1280 and cytosine tothymine at base no. 2225. The amino acid sequence of a polypeptideencoded in SEQ ID NO: 1 is represented by SEQ ID NO: 2.

Example 2 Evaluation of Ability to Suppress Expression of OCLN-ARHGAP26Fusion Protein in a Stomach Cancer Cell Line Expressing OCLN-ARHGAP26Fusion Gene Using ARHGAP26 siRNA, and Evaluation of Viability of theCell Line Under the Condition

After culturing the stomach cancer cell line OKAJIMA that expressesOCLN-ARHGAP26 fusion gene, as shown in Example 1 in RPMI-1640 medium(Wako Pure Chemical Industries, Ltd.) containing 10% bovine serum(Gibco), siRNA was introduced into the cells according to the standardprotocol of the transfection reagent DharmaFECT1 (GE Healthcare).Specifically, the above cancer cells were seeded at 2×10⁵ cells per wellto a 6 well plate (140675, Nunc). 75 pmol of siRNA that targets ARHGAP26(s23013, Life Technologies) and control siRNA (AM4611, LifeTechnologies) were added to the cells (final concentration 75 nM), andthe cells were cultured at 37° C. under an environment of 5% CO₂ for 120h. (hereinafter, the group in which control siRNA was transfected isreferred to as the Control siRNA group, and the group in which siRNAthat targets ARHGAP26 is transfected is referred to as the ARHGAP26siRNA group).

The suppressive effect of OCLN-ARHGAP26 fusion protein by siRNAtreatment was evaluated by the Western blot analysis. Specifically, thecultured cells were dissolved in 350 mM dithiothreitol(Fermentas)-containing Laemmli Sample Buffer (Bio-Rad) to extractprotein. Protein concentration was measured by Protein QuantificationAssay (MACHEREY-NAGEL GmbH & Co. KG). The protein extract was loadedonto a 8% or 12% Poly-Acrylamide gel (Serva) containing SDS (Wako PureChemical Industries, Ltd.) so that 5 μg or 20 μg of protein was loadedonto each lane and gel electrophoresis was performed for 1 h. under acondition of 40 mA. After 80 min of transfer to a PVDF membrane(Millipore Corporation) under a 60 mA condition using TRANS-BLOT SDSEMI-DRY TRANSFER CELL (Bio-Rad), blocking was performed for 2 h. atroom temperature using PBS containing 5% Membrane Blocking Agent (GEHealthcare) (hereinafter referred to as the blocking buffer). Themembrane was shaken in a primary antibody solution of anti-ARHGAP26antibody (HPA035107, Sigma-Aldrich) diluted with a blocking buffer to arate of 1:500 and anti-β-Actin antibody (4967, Cell SignalingTechnology) diluted with a blocking buffer to a rate of 1:3000, andincubated overnight at 4° C. After washing with PBS containing 0.05%Tween 20 (Wako Pure Chemical Industries, Ltd.) (hereinafter referred toas the washing buffer), the membrane was shaken in a secondary antibodysolution of HRP labeled anti-rabbit antibody (P0399, Daco) diluted witha blocking buffer to a rate of 1:3000, and incubated for 1 h. at roomtemperature. After washing with a washing buffer, Pierce Western blotSubstrate Plus (Thermo Fisher Scientific Inc.) was added onto themembrane, and the chemiluminescence on a membrane was detected usingLAS-4000R (Fuji Film). As a result of Western blotting, it was confirmedthat the expression of OCLN-ARHGAP26 fusion protein was suppressed bythe siRNA that targets ARHGAP26 (FIG. 1).

Next, in order to evaluate the effect of the OCLN-ARHGAP26 fusion geneon the viability of the cancer cells, siRNA that targets ARHGAP26 andthe control siRNA were introduced into the stomach cancer cell lineOKAJIMA under the same conditions as shown above. After 24 h., themedium was changed to a RPMI-1640 medium containing 0.5% bovine serum,and the cells were seeded at 1×10³ cells per well to a 96 well plate(167008, Nunc), at 100 μL each, so that cells of each group were seededto 6 wells, and cultured for additional 48 h. at 37° C. under a 5% CO₂environment. Wells containing only RPMI-1640 medium containing 10%bovine serum without cells was prepared as a control (hereinafterreferred to as the medium group). The number of living cells wasmeasured according to the standard protocol of Cell Counting Kit-8(DOJINDO LABORATORIES). Specifically, 10 μL of the reagent was added perwell and the cells were cultured for 4 h. at 37° C. under a 5% CO₂environment, then, the number of living cells was determined bymeasuring an absorbance of 450 nm by a micro plate reader (BioTek).Total 4 wells excluding the maximum and the minimum absorbance values ofeach group were adopted for the analysis. Viability of the Control siRNAgroup and the ARHGAP26 siRNA group was determined by subtracting theabsorbance of the medium group from the absorbance of each group(hereinafter referred to as the correction value), and setting thecorrection value of the Control siRNA group as 100%. Student's t-testwas used for the significance test between Control siRNA group andARHGAP26 siRNA group. Statistical significance was determined when thep-value was given less than 0.05.

Consequently, the viability of cells decreased significantly (FIG. 2)when the expression level of OCLN-ARHGAP26 fusion protein was suppressedby introducing siRNA that targets ARHGAP26 into the stomach cancer cellline OKAJIMA, which is a cell line that endogenously expressesOCLN-ARHGAP26 fusion gene. It was thus found that suppressing theexpression of the fusion gene in cancer cells that endogenously expressthe OCLN-ARHGAP26 fusion gene inhibited the growth of cancer cellsand/or decreased the survival of those cells.

Thus, it was found that the OCLN-ARHGAP26 defined the tumor advancingcapacity of cancer cells.

Example 3 Detection of OCLN-ARHGAP26 Fusion Gene

Total RNA prepared from the stomach cancer cell lines KATO-III(JCRB0611, JCRB cell bank) and HSC-39 (provided from National CancerCenter Japan, Animal Experiment Section) and stomach cancer cell linesNSC-9C, NSC-6C and NSC-16C that were established at the National CancerCenter Japan, Biomarker Search Section, in addition to the stomachcancer cell line OKAJIMA that expresses an OCLN-ARHGAP26 fusion geneshown in Example 1, were reverse-transcribed into cDNA using reversetranscriptase (SuperScriptIII; Life Technologies) and oligo(dT) primer(oligo(dT)20 primer; Life Technologies).

Next, primers of OCLN-ARHGAP26_O5A12_partial fwd02 represented by SEQ IDNO: 7 and OCLN-ARHGAP26_O5A12_partial rev02 of SEQ ID NO: 8 were used toperform PCR (2 min at 94° C., followed by 15 sec. at 94° C., 15 sec. at55° C., and 1 min at 68° C., 30 cycles) using DNA polymerase (AccuPrimeTaq DNA Polymerase; Life Technologies) and cDNA obtained above as atemplate (200 ng when converted to total RNA). Likewise, to confirm theequal amount of cDNA template in the reactions, primers of ACTB_F2represented by SEQ ID NO: 9 and ACTB_R2 represented by SEQ ID NO: 10were used to perform PCR (2 min at 94° C., followed by 15 sec. at 94°C., 15 sec. at 55° C., and 1 min at 68° C., 25 cycles) using the sameDNA polymerase as above. Electrophoresis was performed with 2% agarosegel (Lonza) after PCR reaction, and about 500 bp PCR product wasamplified only at the stomach cancer cell line OKAJIMA which was alreadyconfirmed the expression of OCLN-ARHGAP26 fusion gene (FIG. 3). The PCRproduct amplified by the aforementioned primer set was 511 bp accordingto the nucleotide sequence of the fusion gene identified in Example 1.Therefore, it was shown that it is possible to detect the fusion geneexpressed in cancer cells with PCR method.

Example 4 Evaluation of the Viability of Stomach Cancer Cell LineExpressing OCLN-ARHGAP26 Fusion Gene Under Suppression of OCLN-ARHGAP26Fusion Protein Expression

The stomach cancer cell line OKAJIMA that expresses OCLN-ARHGAP26 fusiongene shown in Example 1 was cultured in RPMI-1640 medium containing 10%bovine serum, then the target siRNA was introduced according to thestandard protocol of a transfection reagent DharmaFECT1 (T2001-03, GEHealthcare). Specifically, the aforementioned cancer cells were seededat 2×10⁵ cells per well in a 12 well plate (3815-012, AGC TECHNO GLASSCo., Ltd.), then two siRNAs targeting OCLN (Ambion s9814 and s458015(both by Life Technologies)) and three siRNAs targeting ARHGAP26 (Ambions23013, s23015 (both by Life Technologies) and SI03077690 (Qiagen)) wereadded to the cells at 75 pmol each (final concentration 75 nM), andcultured for 72 h. at 37° C. under a 5% CO₂ environment. At the sametime, AllStars Negative Control siRNA (1027280, Qiagen) was similarlyadded to the cells as a negative control and siRNA (SI02653770, Qiagen)that targets KIF11 was similarly added to the cells as a positivecontrol of apoptosis, and the cells were cultured for 72 h. at 37° C.under a 5% CO₂ environment. Among the siRNAs targeting ARHGAP26, s23015was expected to target only wild-type ARHGAP26 and not OCLN-ARHGAP26based on its sequence.

The suppressive activity of siRNA to the OCLN-ARHGAP26 fusion proteinexpression was evaluated by Western blot. Specifically, the culturedcells were dissolved in the Cell Lysis Buffer (9803, Cell SignalingTechnology, Japan) of a 1× concentration comprising a protease inhibitorcocktail (25955-11, Nacalai) and a Halt Protease and PhosphataseInhibitor Cocktail (78441, Thermo Fisher Scientific) each at a 1/100amount to extract protein. The protein concentration was measured by BCAProtein Assay Kit (23227, Thermo Fisher Scientific). The protein extractwas loaded onto a NuPAGE Novex 4-12% Bis-Tris Gel (NP0322BOX, ThermoFisher Scientific) so that there will be 4 μg of target protein extractper lane, and gel electrophoresis was performed for 40 min under acondition of 180 V, followed by transferred to a PVDF membrane(162-0176, Bio-Rad) under a condition of 120 min, 190 mA using a twinmini buffer transfer device (BE-351W, Biocraft). Then, the membrane wasblocked with PVDF Blocking Reagent for Can Get Signal (NYPBR01, TOYOBO)for 1 h. at room temperature. The membrane was shaken in a primaryantibody solution of anti-ARHGAP26 antibody (HPA035107, AtlasAntibodies) and anti-β-Actin antibody (4967S, Cell Signaling Technology)respectively diluted with Can Get Signal Immunoreaction EnhancerSolution1 (NKB-201, TOYOBO) to a rate of 1:1,000, and reacted overnightat 4° C. After washing with Tris buffer solution containing0.2% Tween 20(hereinafter referred to as a washing buffer), the membrane was shakenin a secondary antibody solution of HRP labeled anti-rabbit antibody(NA9340, GE Healthcare) diluted with Can Get Signal ImmunoreactionEnhancer Solution2 (NKB-301, TOYOBO) to a rate of 1:10,000, and reactedfor 1 h. at room temperature. After washing with a washing buffer, theECL Prime Western Blotting Detection Reagent (RPN2232, GE Healthcare)was added onto the membrane and the chemiluminescence signal wasdetected from the membrane using LAS-4010 (GE Healthcare). As a resultof Western blotting experiment, it was confirmed that the expression ofOCLN-ARHGAP26 fusion protein was suppressed by two OCLN siRNAs and twoARHGAP26 siRNAs, other than s23015.

To evaluate the impact of OCLN-ARHGAP26 fusion gene on the viability ofthe cancer cells, transfection of siRNAs targeting OCLN, ARHGAP26 andthe control siRNA to the stomach cancer cell line OKAJIMA was performedunder the same condition as mentioned above. After 24 h., the medium waschanged to RPMI-1640 medium containing 10% bovine serum, and the cellswere seeded at 1×10³ cells per well to a 96 well plate (3860-096, AGCTECHNO GLASS Co., Ltd.), at 100 μL each, so that each group was seededin 3 wells, and wells added only RPMI-1640 medium containing 10% bovineserum without any cells seeded thereto (hereinafter referred to as themedium group), and the cells were subjected to further culturing at 37°C. under an environment of 5% CO₂ for 48 h. (hereinafter referred to asday 3) and 120 h. (hereinafter referred to as day 6). Further, as acontrol for normalization, plates in which cells are only seeded and notcultured were prepared (herein after day 1). The number of living cellswas determined according to the standard protocol of Cell Titer GloLuminescent Cell Viability Assay (G7571, Promega Corp.) at the timepoint given above. Specifically, 100 μL of a reagent was added per welland the cells were incubated for 10 min at room temperature, then, thenumber of living cells was determined by measuring the luminescence by amicro plate reader Infinite M1000 (Deccan). The viability of the siRNAtreated cell group of each time point was obtained by subtracting theluminescence of the medium group from the luminescence of each group(hereinafter referred to as the correction value), and setting thecorrection value of the siRNA treated group on day 1 as 100%.

Consequently, the viability of cells decreased significantly (FIG. 4) bythe transfection of siRNA targeting a fusion gene to suppress theexpression of the OCLN-ARHGAP26 fusion protein in the stomach cancercell line OKAJIMA, which is a cell line that endogenously expressesOCLN-ARHGAP26 fusion gene (FIG. 4). It was thus found that suppressingthe expression of the fusion gene in cancer cells that endogenouslyexpress the OCLN-ARHGAP26 fusion gene inhibited the growth of cancercells and/or decreased the survival of those cells.

Therefore, it was shown that OCLN-ARHGAP26 is involved in the cancercell's ability to expand tumor.

INDUSTRIAL APPLICABILITY

The detection method of the present invention is a method for detectinga fusion gene composed of the OCLN gene and the ARHGAP26 gene, and it isuseful as a method for detecting and diagnosing cancer in a subject.Further, the primer set and the detection kit of the present inventionmay be used in a method of the present invention.

1-14. (canceled)
 15. A method of treating cancer in a subject, themethod comprising administering to the subject a pharmaceuticalcomposition comprising a substance for suppressing the expression of afusion protein of occludin (OCLN) and Rho GTPase activating protein 26(ARHGAP26), wherein the fusion protein is a polypeptide according toeither (1) or (2): (1) a polypeptide that comprises an amino acidsequence having no less than 90% identity with an amino acid sequence ofSEQ ID NO:2; (2) a polypeptide that comprises an amino acid sequence ofSEQ ID NO:2, or a polypeptide that comprises an amino acid sequence ofSEQ ID NO: 2, in which 1 to 10 amino acids are deleted, substituted,inserted and/or added, thereby treating cancer in the subject.
 16. Themethod of claim 15, wherein the fusion protein is a polypeptidecomprising an amino acid sequence having no less than 90% identity withan amino acid sequence of SEQ ID NO:2.
 17. The method of claim 15,wherein the fusion protein is a polypeptide comprising an amino acidsequence of SEQ ID NO:2, or the polypeptide comprises an amino acidsequence of SEQ ID NO:2, in which 1 to 10 amino acids are deleted,substituted, inserted and/or added.
 18. The method of claim 15, whereinthe fusion protein is a polypeptide consisting of an amino acid sequenceof SEQ ID NO:2.
 19. The method of claim 15, wherein the cancer istreated by inhibiting the growth of cancer cells and/or decreasing thesurvival of cancer cells in the subject.
 20. The method of claim 15,wherein the substance for suppressing the expression of the fusionprotein of OCLN and ARHGAP26 is an siRNA targeting ARGHAP26.
 21. Themethod of claim 15, wherein the cancer is stomach cancer.